The Molecular Mechanism Of PbrmiR397a And PbrMYB169 Regulates Lignin Biosynthesis In Stone Cell Of Pear Fruit

Stone cell is unique in pear flesh,and is also one of important factors affecting the taste and processing quality of pear fruit.The fruits of main pear varieties have high stone cells content,rough flesh and taste which seriously affect the fruit quality and economic value.Previous research had reported that stone cells belong to a type of brachysclereid,and lignin deposition in secondary cell wall is one of important reasons leading to harden stone cell.To date,lignin studies almost focus on model plant,while the molecular mechanism regulating lignification is poorly understood in fruit stone cells.In this study,based on the analyses of microRNA sequencing data and MYB genome family members identification,we found key candidate genes,PbrmiR397a and PbrMYB169,which may regulate lignification during stone cell formation.The molecular mechanism and gene function analysis were carried out on the two candidate genes.The results are as follows:1.Stone cell content accumulated rapidly in the fruit from 21 to 49 days and remained stable from 49 DAF.Stone cells and lignin will not be degraded after their formation but be maintained in fruit flesh.Through the analyses of pear flesh microRNA sequencing and target gene prediction,PbrmiR397a targeted multiple laccase(LAC)genes but their function in stone cell formation is unknown.38 laccase(LAC)genes were identified from pear genome but.Based on the results of conserved domain and phylogenetic analyses,LACs were grouped into six clades.Six of them expressed abundantly at 21 and 35 DAF,after which the expression decreased rapidly.The subcellular localization analysis showed these LAC proteins are specifically located in cell wall,which is in accordance with the position of the biosynthesis of lignin monomer.Both 5’-RACE and reporter gene analysis verified that all LACs were targets of PbrmiR397a,and PbrmiR397a expression contrasted with that of PbrLACs at 21 and 35 DAF.The results supported a regulatory role of PbrmiR397a in suppressing these LACs.Transient overexpression of PbrmiR397a and simultaneous silencing of three LAC genes reduced the lignin content in pear fruit.The biomass of T2 homozygous transgenic tobacco showed no significant differences from that of wild type control plants.However,the T2 plants showed 18%to 29%reduction in lignin content compared with WT control plants.Even there was no change in the ratio of S/G lignin monomers compared with WT plants.The vascular tissue of transgenic plants had highly reduced numbers of vessel elements and thinner xylem regions.The secondary wall thickness of transgenic PbrmiR397a lines was also significantly lower than that of WT plants.Through q-PCR analysis,nine LACs were down-regulated by 28-90%in the PbrmiR397a transgenic line compared with the WT.2.Based on the correlation results between re-sequencing and stone cell content analyses data,six SNPs in the promoter region of the PbrmiR397a precursor gene were found.Through Fisher’s exact test analysis,P-value of each SNP were significantly less than 0.05.A phylogenetic tree based on the upstream sequences of PbrmiR397a classified all 30 HG varieties into one clade and all 30 LG varieties into another clade.SNP marker primers were designed for screening varieties and crossing progenies which have low stone cell content.Using the PlantCARE database for analyses of conserved promoter elements,we found two of the SNPs will change hormone-responsive elements in the promoter region of PbrmiR397a in the HG and LG.The dual-luciferase reporter assay results showed that the TCA-element caused by the change of SNP#7(G>A)in the LG varieties could be activated by SA.In pear fruit,the expression level of PbrmiR397a was significantly lower in the 15 HG varieties than that in 15 LG varieties.In contrast,the expression level of PbrLACs was higher in the 15 HG varieties than that in the 15 LG varieties.Based on these results,we propose that the alterations to cis-elements caused by SNP#7 may be responsible for the different expression levels of PbrmiR397a between the HG and LG varieties and eventually results in the change of pear flesh stone cell content.3.Through screening of the whole genome MYB gene family,we found the candidate gene PbrMYB169 in relation to stone cells formation of pear fruit.Protein sequence of PbrMYB169 contains typical R2-and R3-type MYB repeat domain,and clusters with AtMYB85 which is lignin-specific activators in Arabidopsis.The subcellular localization analysis showed this PbrMYB169 protein was localized to nucleus.Combining co-expression network and expression profiles analysis,4CL1、4CL2、C3H1、C3H2、CAD、CSE、CCOMT2、CCR1、CCR2、HCT2、HCT4、LAC1、LAC2、LAC15、LAC18 genes were selected as vital candidate genes involving in catalysing lignin biosynthesis during the formation of stone cells in pear fruit.Dual luciferase assay demonstrated that recombinant PbrMYB 169 protein significantly induced transactivation of the promoter of C3H1、CCR1、CCOMT2、CAD、4CL1、4CL2、HCT2、LAC18 via binding to AC elements.Overexpression of PbrMYB 169 in transgenic Arabidopsis plants resulted in development arrest,stronger lignin deposition and more thickened secondary cell walls in vessel elements compared with wild-type plants.And expression profiles assay indicated a substantial induction in the expression of genes involved in monolignol biosynthesis.